Megalin Functions as an Endocytic Sonic Hedgehog Receptor

Robert A. McCarthy1, Jeremy L. Barth1, Mastan R. Chintalapudi, Christian Knaak

and W. Scott Argraves2

Running title: Megalin as a N-Shh receptor

Key words: gp330, LRP2, patched, zebrafish

Medical University of South Carolina

Department of Cell Biology

171 Ashley Avenue

Charleston, SC 29425-2204 USA

1These authors contributed equally to this work.

2To whom all correspondence should be addressed. E-mail: argraves@musc.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 18, 2002 as Manuscript M201933200 by guest on A

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Summary

Embryos deficient in the morphogen sonic hedgehog (Shh) or the endocytic receptor

megalin exhibit common neurodevelopmental abnormalities. Therefore, we have investigated

the possibility that a functional relationship exists between the two proteins. During embryonic

development megalin was found to be expressed along the apical surfaces of neuroepithelial

cells and was co-expressed with Shh in the ventral floor plate of the neural tube. Using ELISA,

homologous ligand displacement and surface plasmon resonance techniques it was found that

the amino-terminal fragment of Shh (N-Shh) bound to megalin with high affinity. Megalin

expressing cells internalized N-Shh through a mechanism that was inhibited by antagonists of

megalin, namely, RAP and megalin antibodies. Heparin also inhibited N-Shh endocytosis,

implicating proteoglycans in the internalization process as has been described for other megalin

ligands. Use of chloroquine to inhibit lysosomal proteinase activity showed that N-Shh

endocytosed via megalin was not efficiently targeted to the lysosomes for degradation. The

ability of megalin-internalized N-Shh to bypass lysosomes may relate to the finding that the

interaction between N-Shh and megalin was resistant to dissociation with low pH. Together

these findings show that megalin is an efficient endocytic receptor for N-Shh. Furthermore, they

implicate megalin as a new regulatory component of the Shh signaling pathway.

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Introduction

Sonic hedgehog (Shh) is a secreted signaling molecule that is expressed in spatially

restricted patterns during embryonic development. Shh signaling has been shown to regulate a

wide range of developmental patterning events in Drosophila and vertebrates involved in lung

(1), nervous system (2), eye (3), midbrain (4), forebrain and facial morphogenesis (5, 6). During

early vertebrate development, Shh signaling at the midline leads to patterning of the ventral

neural tube and adjacent somites. Mice lacking Shh activity have anomalies of midline

structures such as the notochord and floorplate of the early brain (7). Later these mice display

an absence of ventral neuronal cells and cranial motor neurons (8). The result of errant Shh

signaling in humans has been directly linked to basal cell carcinoma (9, 10) and

holoprosencephaly (HPE) (11).

Post-translational modification of the 45 kDa Shh polypeptide produces an ~19-kDa

amino-terminal fragment, designated N-Shh which has palmitic acid and cholesterol moieties

covalently coupled to its amino and carboxy termini, respectively (12-14). N-Shh is secreted

and represents the biologically active form of the protein, capable of initiating signaling. The

current model for Shh signaling involves a pair of multiple-pass plasma membrane proteins,

patched (Ptc or Ptc-1) and smoothened (Smo) (reviewed in (15)): Ptc functions as the Shh-

binding subunit/receptor and Smo as the signal transducing subunit. When bound to Smo, Ptc

acts as a repressor of Smo signaling activity. Following N-Shh interaction with Ptc, bound Ptc

releases from Smo and de-represses the signaling activity of Smo. The expression of Ptc-1,

Gli-2, HNF3beta, Nkx2.2 and netrin-1 have been shown to be activated by Shh, and genes

including Pax-3, Gli-3 and ephrin A5 have been shown to be suppressed by Shh (16, 17).

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Megalin (also known as gp330 and LRP-2) is an endocytic receptor belonging to the low

density lipoprotein (LDL) receptor family (18). The receptor is expressed on apical surfaces of

numerous epithelia where it functions to mediate endocytosis of ligands, targeting them for

lysosomal degradation or transcytosis (18). Mice deficient in the expression of megalin

demonstrate the critical neurodevelopmental role for this protein (19). These mice display

numerous craniofacial abnormalities including absence of olfactory bulbs, absence of the corpus

callosum and fusion of forebrain hemispheres, collectively an HPE phenotype (19). During

development, megalin-deficient embryos (9.5 dpc) have pronounced cell death in several

structures including cranial nerves, neural crest and the optic vesicle (19). The spectrum of

defects that constitute the megalin-deficient phenotype suggests that megalin expression is

required for normal viability of the neural epithelium at an early embryonic stage.

The phenotype of megalin-deficient mice suggests a role for megalin in regulating cell

fate specification in the patterning of the neural tube and is consistent with phenotypes

observed in mice deficient in Shh and the Shh signal transducer, Smo (8, 20). For example,

Shh-deficient embryos lack cranial motor neurons (8). Inhibition of Shh signaling in the neural

tube has been shown to result in extensive apoptosis of neural epithelial cells (21). Shh has

also been shown to regulate proliferation and inhibit differentiation of central nervous system

(CNS) precursor cells (22). Smo mutants also display neural tube-related defects including

increased apoptosis of cells within the neural tube, absence of secondary motor neurons,

synopthalmia and ventral forebrain defects (20, 23). The shared aspects of the megalin-, Shh-

and Smo-deficient phenotypes suggest that Shh and megalin impact common mechanisms that

underlie CNS development. Here we report findings from experiments directed at determining

whether a functional relationship exists between megalin and Shh.

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Experimental Procedures

Cells. MSV-transformed Brown Norway rat yolk sac cells (BN cells) were provided by Dr. Pierre

Verroust (Hospital Tenon, Paris, France). Mouse embryonal teratocarcinoma F9 cells (ATCC

CRL1720) were differentiated by treatment with retinoic acid and dibutyryl cyclic AMP

(Bt2/cAMP) for 6 days as previously described (24). C3H10T1/2 (ATCC CCL-226) were obtain

from the American Type Culture Collection (Manassas, VA).

Antibodies. Rabbit polyclonal and mouse monoclonal antibodies to megalin, rb6286 and 1H2,

have been described previously (25). Rabbit anti-megalin IgGs were purified by protein-G-

Sepharose and megalin-Sepharose chromatography (26). Mouse monoclonal RAP antibody

7F1 has been described previously (27). Mouse monoclonal N-Shh antibody 5E1 IgG was

isolated from the conditioned culture medium of a hybridoma cell line obtained from the

Developmental Studies Hybridoma Bank (The Johns Hopkins University School of Medicine and

the University of Iowa). Goat anti-glutathione-S-transferase (GST) was obtained from

Amersham Pharmacia Biotech (Piscataway NJ). Fluorescein isothiocyanate (FITC) and

indocarbocyanine (Cy3) labeled secondary IgGs were purchased from Jackson

ImmunoResearch Labs, Inc. (West Grove, PA).

Proteins. Megalin was purified from porcine kidney as described previously (28). Human RAP

was expressed in bacteria and purified as described by Kounnas et al. (29). Recombinant

murine N-Shh (residues 25-198) was obtained from R&D Systems (Minneapolis, MN). A

plasmid construct was created to express GST-N-Shh fusion protein in bacteria. Briefly, this

involved using RT-PCR to generate a cDNA encoding amino acids 20-198 of Shh from cDNA

template prepared from 9.5 dpc mouse embryo RNA. The Shh cDNA fragment was inserted

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into the bacterial expression vector pGEX-2TK (Amersham Pharmacia Biotech) such that the

resulting plasmid encoded a fusion protein composed of GST followed by a thrombin cleavage

site (LVPRGS), a five amino acid phosphorylation target site (RRASV) and the N-Shh

polypeptide. The construct was transformed into BL21 bacteria and the fusion protein was

isolated using glutathione-Sepharose affinity chromatography. Recombinant GST was

produced from cells transformed with the empty pGEX-2TK vector. Both recombinant protein

preparations were adsorbed onto a Detoxigel Endotoxin Removing Gel (Pierce, Rockford, IL).

The biological activity of GST-N-Shh was assayed in C3H10T1/2 cells using the method of

Williams et al. (30).

Radiolabeling of GST-N-Shh and GST-RAP. GST-N-Shh and GST-RAP were labeled with [γ -

32P]-ATP using heart muscle kinase (HMK) based on manufacturer recommendations (Sigma)

and the protocol of Stefansson et al. (31). Briefly, 50 µg of protein was incubated with 125 units

of HMK in 1X HMK buffer (20 mM PIPES, pH 6.5, 1 mM DTT, 20 mM NaCl, 12 mM MgCl2) plus

0.1% denatured BSA and 50 µCi [γ -32P]-ATP (Amersham Pharmacia Biotech) for 1h. Labeled

fusion protein was purified by size exclusion chromatography using PD-10 columns. Typical

specific activities were 0.5-2 x 108 cpm/nmol.

Wholemount embryo immunolabeling. Zebrafish were maintained and embryos collected by

standard methods (32). Embryos were fixed for 15 min in 4% paraformaldehyde in phosphate-

buffered saline (PBS). Embryos were washed two times in PBS, permeabilized by washing

three times in PBS containing 0.1% saponin (sap/PBS) at 37°C, and blocked for 30 min (37°C)

in PBS containing 5% goat serum and 0.1% saponin (gs/sap/PBS). Embryos were incubated

with primary antibody (5 µg/ml) in gs/sap/PBS first for 1 h at 37°C, then overnight at 4°C and

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finally for an additional 1 h at 37°C with rocking. Embryos were washed three times with

gs/sap/PBS at 37°C, and incubated in Cy3-coupled goat anti-mouse or anti-rabbit secondary

antibody (Jackson ImmunoResearch Laboratories) 1.5 µg/ml in gs/sap/PBS for 1 h at 37°C.

Samples were washed three times in gs/sap/PBS, dehydrated in methanol, followed by clearing

in Murray’s Clear (1:2 benzylalcohol: benzylbenzoate). Laser scanning confocal microscopy

(LSCM) was performed using a BioRad MRC-1400 confocal microscope and BioRad

LaserSharp2000 software.

Immunoblotting and ligand overlay assay. Detergent extraction of cells and immunoblot

detection of megalin was performed as described previously (24). RAP ligand blot overlay

assay was performed as described by Battey et al. (27).

Solid-phase binding assays. Enzyme-linked immunosorbent assay (ELISA) was performed

essentially as described previously (33). Briefly, varying concentrations of N-Shh (R&D

Systems) or GST-N-Shh in 150 mM NaCl, 50 mM Tris pH 7.4, 3% nonfat milk, 0.05% Tween 20

were incubated for 1 h at 37oC in microtiter wells coated with megalin (3 µg/ml). Bound N-Shh

was detected using the monoclonal antibody 5E1, sheep anti-mouse IgG-horse radish

peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ) and the chromogenic substrate

3,3',5,5'-tetramethyl-benzidine (TMB) (Kirkegaard & Perry, Gaithersburg, MD).

For homologous ligand competition assays, [32P]-labeled GST-N-Shh (1 nM) was

incubated in microtiter wells coated with megalin (3 µg/ml) in the presence of increasing

concentrations of unlabeled competitor (GST-N-Shh or RAP). All other conditions were similar

to those described in Williams et al. (34). The algorithm Ligand (35) within SigmaPlot 7.101 was

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used to analyze the competition data and to determine dissociation and inhibition constants (Kd)

for receptor-ligand interactions.

Kinetic analysis of N-Shh-megalin binding. Kinetic analysis of the interaction of GST-N-Shh

with purified megalin was performed using surface plasmon resonance (SPR) measurements

made on a BIAcore 3000 instrument (BIAcore, Inc., Uppsala, Sweden). BIAcore sensor chips

(type CM5) were activated with a 1:1 mixture of 0.2 M N-ethyl-N’-(3-dimethylaminopropyl)

carbodiimide and 0.05 M N-hydroxysuccinimide in water. Megalin (50 µg/ml, 83 nM in 10 mM

sodium acetate, pH 4.8) was immobilized onto a CM5 sensor chip using the amine-coupling kit

as described by the supplier, BIAcore. Unreacted sites were blocked with 1M ethanolamine, pH

8.5. The SPR signal from immobilized megalin generated BIAcore response units ranging from

20,000-28,000. Control flow cells were activated and blocked in the absence of protein.

Binding was evaluated over a range of GST-N-Shh concentrations (25-500 nM) in 150 mM

NaCl, 0.005% polysorbate 20 and 100 mM Hepes (pH 7.4) plus and minus 1 mM CaCl2 at 25°C.

Binding of GST-N-Shh to megalin-immobilized flow cells was corrected for binding to control

flow cells. Binding data were fitted to a 1:1 (Langmuir) binding model using the BIAevaluation

3.1 software (BIAcore).

To evaluate the effect of pH on the dissociation of megalin-ligand complexes, GST-N-

Shh or RAP (each at 3 µM in 100 mM Hepes pH 7.4, 150 mM NaCl [HBSN]) were passed at 10

µl/minute for 2 minutes over sensor chips containing immobilized megalin. Subsequently,

protein-free HBSN or sodium acetate buffer pH 4.5 (sodium ion concentration adjusted to 150

mM) was applied for 5 minutes. The kinetic dissociation profiles obtained under neutral and

acid pH conditions were used to calculate off-rates (koff) using the BIAevaluation 3.1 program.

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Between replicate experiments the chip surface was regenerated with a 10-sec pulse of 10 mM

glycine, pH 2.2 at 5 µl/min.

Confocal microscopic analysis of N-Shh uptake. BN cells were plated at 0.25 x105 cells/cm2

in eight-well chamber slides (Nunc Nalge, Naperville, IL) in Eagles Minimal Essential Medium

(MEM), containing 10% fetal bovine serum, nonessential amino acids, 100 units/ml penicillin

and 100 µg/ml streptomycin (complete medium). Cells were grown for 16 h at 37°C, 5% CO2

and the medium was replaced with serum-free medium (MEM, nonessential amino acids, 100

units/ml penicillin and 100 µg/ml streptomycin, insulin, transferrin and selenic acid). After a 1.5

h incubation the medium was replaced with serum-free medium containing 1.5% BSA and either

GST-N-Shh (20 nM) or GST (20 nM) plus or minus competitors and cultured for 2 h.

Competitors included RAP (1 µM), GST (1 µM).

For immunological detection GST-N-Shh- and GST-treated cells were rinsed in

Dulbecco’s phosphate buffered (DPBS) saline, pH 7.4 (DPBS), fixed for 20 min in 3.7%

paraformaldehyde with 0.2% Triton-X100 in DPBS, and then rinsed with DPBS. Cells were

incubated with 2% BSA in DPBS for 1 h and treated with goat anti-GST IgG (Amersham) at 1

µg/ml in 2% BSA/DPBS for 1 h, then FITC-donkey anti-goat IgG at 3 µg/ml in DPBS for 1 h, and

then rinsed in DPBS. For nuclear staining, cells were treated with RNase A (100 µg/ml) for 20

min at 37°C, rinsed in DPBS, and then treated with TOTO-3 (Molecular Probes, Eugene, OR) at

1 µg/ml in DPBS for 10 min at 37°C. Cells were rinsed in DPBS, mounted in Vector Shield

mounting solution (Vector Laboratories, Burlingame, CA) and then examined using LSCM.

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Cellular internalization and degradation assays. BN were seeded into wells of 24-well plates

at 0.5 x105 cells/cm2 and grown for 16 h at 37°C, 5% CO2 in complete medium and then for 1.5

h in serum free medium. Medium was then replaced with serum-free medium plus 1.5% BSA

and [32P]-labeled GST-N-Shh (3 nM) with or without indicated agents (i.e., RAP, IgG or heparin),

and cells were grown for 2-6 h. For experiments measuring the effect of chloroquine treatment,

chloroquine was added at 0.1 mM concomitantly with the addition of radiolabeled ligands, and

uptake allowed to proceed for 6 h. Quantification of the amount of bound, internalized and

degraded ligands was performed as described previously (36). Radioactivity in the cell medium

that was soluble in 10% trichloroacetic acid (TCA) was taken to represent degraded ligand.

Total ligand degradation was corrected for the amount of degradation that occurred in

radioligand-containing medium in the absence of cells. To determine the amount of [32P]-ligand

that was bound and internalized, cells were washed three times with DPBS and then treated

with serum-free medium containing 0.5 mg/ml trypsin, 0.5 mg/ml proteinase K (Sigma) and 0.5

mM EDTA for 2-4 minutes at 4°C. The cell suspension was then centrifuged at 6000 x g for 4

min and the amount of radioactivity in the supernatant was taken to represent the bound fraction

while the amount in the cell pellet was taken as the internalized fraction.

Uptake experiments with differentiated and control F9 cells were performed as above

with the exception that cells were seeded at 1.0 x105 cells/cm2, and the growth medium was

DMEM, 10% FBS (or insulin, transferrin and selenic acid) containing 100 units/ml penicillin and

100 µg/ml streptomycin.

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Results

Neurodevelopmental expression of megalin. Despite indications that megalin is critical to

neurodevelopment (19), little was known about the expression of the receptor during early

development. Laser scanning confocal microscopic analysis of 16-hour zebrafish embryos

revealed that megalin was prominent in the floorplate of the neural tube (arrow, Fig. 1A) and the

apical surface of the optic cup (arrowhead, Fig. 1A). By 24-hours, megalin expression was

detected in cells of the ventral floorplate (arrow, Fig. 1B) and on the apical surface of cells lining

the lumen of the neural tube (arrowhead, Fig. 1B). At 33-hours, ventral floorplate expression

persisted and megalin was also extensively expressed on cells comprising the lumenal surfaces

of the forebrain and midbrain (arrowhead, Fig. 1D) with strong expression at the midbrain-

hindbrain border (arrowhead, Fig. 1C). At the base of the midbrain, intense staining for megalin

was seen at the most anterior extent of the floorplate (arrow, Fig. 1D). Outside the central

nervous system, megalin was detected on the apical surfaces of cells lining the lumen of the otic

vesicle of the developing ear (arrowhead, Fig. 1E). In the area of the developing mouth of 48-

hour embryos, megalin was distributed medially and laterally in the frontonasal and maxillary

processes, respectively (Fig. 1G). These findings demonstrate that early embryonic expression

of megalin occurs at specific organizing centers for morphogenesis including the ventral neural

tube, optic and otic vesicles and orofacial regions.

Many of the observed embryonic sites of megalin expression were the same as those

known to express Shh including ventral floorplate, eye, otic vesicle and frontonasal process (2-

5, 37). A notable exception was the absence of megalin expression in the notochord

(arrowheads, Fig. 2A and B). Also, megalin expression in the neural tube extended more dorsal

than Shh (inset, Fig. 2 and Fig. 1B), detected in areas of the neural tube known to express the

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receptors for Shh, Ptc-1 and -2 (38). Taken together, megalin is expressed in tissues that

express Shh or in adjacent tissues regulated by Shh signaling. These observations support the

possibility that a functional relationship exists between megalin and Shh during early

neurodevelopment.

Megalin is a N-Shh-binding receptor. The similarity of megalin- and Shh-null phenotypes and

the early embryonic distribution of megalin in relation to sites of Shh production led us to

investigate whether megalin and N-Shh were capable of directly binding to one another.

Enzyme-linked immunosorbant assay (ELISA) showed that a recombinant GST-N-Shh (Fig. 3)

and a commercial preparation of N-Shh bound to purified megalin with similar apparent affinities

(Fig. 4A). Binding between GST-N-Shh and megalin was also tested using a homologous

ligand competition assay. [32P]-GST-N-Shh bound to megalin and the binding was inhibited in a

dose dependent manner by the addition of unlabeled GST-N-Shh (Fig. 4B). A dissociation

constant (Kd) of 81.3 nM was obtained from fitting the data to a single site model using the

Ligand algorithm. Binding of [32P]-GST-N-Shh to megalin was also inhibited by RAP, a well-

established antagonist of megalin-ligand interaction (39). Interestingly, the RAP competition

data could best be fit to a two-site model with inhibition constants (Ki) of 3.0 and 2341.9 nM.

One interpretation for these findings is that RAP binds to megalin at multiple sites and that one

of these binding interactions is a stronger inhibitor of N-Shh binding to megalin. Such an

interpretation is consistent with the fact that the megalin family member, LRP, has multiple RAP

binding sites (34).

Binding of Shh to megalin was also evaluated using surface plasmon resonance (SPR).

As shown in Figure 5, GST-N-Shh bound to megalin immobilized on a sensor chip. GST alone

displayed no measurable binding to megalin (data not shown). Optimal fitting of SPR data

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obtained from measuring the binding of various concentrations of GST-N-Shh to immobilized

megalin were best achieved using a single class binding site model. As a result, an affinity

constant (KD) of 21 nM (n = 7, chi2 of fit <10) was determined for GST-N-Shh binding to megalin

in the presence of calcium. Recombinant N-Shh cleaved with thrombin to remove the amino-

terminal GST moiety and commercial N-Shh were both found to bind megalin immobilized on a

sensor chip with affinities similar to those observed for the fusion protein (data not shown).

Megalin mediates endocytosis of N-Shh. The role of megalin in mediating endocytosis of N-

Shh was next evaluated. As shown in Figure 6, confocal analysis of BN cells cultured in the

presence of GST-N-Shh showed intracellular GST-N-Shh staining in a punctate pattern

consistent with vesicular localization. Cells incubated with GST showed little to no intracellular

staining (Fig. 6A). When BN cells were cultured in the presence of both GST-N-Shh and RAP,

little if any intracellular staining was observed (Fig. 6A). Instead, RAP treated cells displayed

punctate foci of staining located on the cell periphery. This staining pattern is consistent with a

plasma membrane or pericellular localization. Therefore, when megalin activity is abrogated, N-

Shh appears to bind to pericellular matrix or cell surface and uptake is blocked.

We subsequently evaluated the ability of BN cells to mediate endocytosis of radiolabeled

N-Shh. As shown in Figure 7A, BN cells internalized [32P]-GST-N-Shh. The uptake of [32P]-

GST-N-Shh could be blocked by either RAP or antibodies to megalin. The observed inhibitory

effects support the interpretation that megalin mediates N-Shh endocytosis. Furthermore,

inhibition by megalin antibodies alleviates a concern that the inhibitory effects of RAP might not

have been megalin-specific. In this regard it is also important to note that we established

megalin is the only detectable RAP-binding member of the LDLR family in BN cells (Fig. 6B).

Therefore RAP can be considered a specific inhibitor of megalin in BN cells.

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Uptake of GST-N-Shh was also evaluated in murine F9 cells. F9 cells express little or no

megalin but can be differentiated with retinoic acid and Bt2/cAMP causing induced megalin

expression and decreased expression of other LDLR family members (24). As shown in Figure

7B, differentiated cells exhibited an increased capacity to internalize [32P]-GST-N-Shh. RAP

effectively inhibited internalization of [32P]-GST-N-Shh by differentiated F9 but had little effect on

the relatively low level of internalization in undifferentiated cells. These findings further support

the interpretation that megalin mediates endocytosis of N-Shh.

N-Shh endocytosis involves proteoglycans. In light of the fact that cell surface

proteoglycans have been implicated as partners with megalin and other LDLR family members

in the uptake of numerous ligands (18) we investigated their possible involvement in N-Shh

endocytosis. Heparin was an effective inhibitor of the uptake of [32P]-GST-N-Shh by both BN

cells and differentiated F9 cells (Figs. 8A and 8B). This suggests the involvement of cell

surface proteoglycans in the process of N-Shh endocytosis.

N-Shh is not efficiently targeted to lysosomes by megalin. One well-characterized

consequence of megalin-mediated endocytosis is targeting of ligands to the lysosome for

degradation. Inhibition of lysosomal proteinase activity using the drug chloroquine did not inhibit

[32P]-GST-N-Shh degradation in BN cells (Fig. 9). By contrast, in control experiments

chloroquine efficiently inhibited the degradation of [32P]-RAP (Fig. 9), a megalin ligand that is

targeted to the lysosomes following megalin-mediated endocytosis. Interestingly, there was a

significant level of chloroquine-insensitive N-Shh degradation suggesting that degradation of N-

Shh may occur extracellularly.

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Evaluation of lowered pH on dissociation of the N-Shh-megalin complex. The effect of low

pH on the dissociation of the N-Shh-megalin complex was evaluated by SPR on a BIAcore

instrument. Little difference was evident in the dissociation rate constant (koff) for the N-Shh-

megalin interaction under acidic versus neutral pH conditions, 1.3 x 10-3 s-1 and 1.28 x 10-3 s-1,

respectively (Fig. 10A). By contrast, dissociation of the RAP-megalin complex increased ~3-

fold from 3.1 x 10-3 s-1 under neutral pH conditions to 8.36 x 10-3 s-1 under acidic pH conditions

(Fig. 10B). These findings indicate that the N-Shh-megalin interaction is resistant to

dissociation by acidic pH as low as 4.5 and suggest that N-Shh may not readily dissociate from

megalin under acidic pH within endosomes.

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Discussion

Here we establish that a functional relationship exists between the endocytic receptor

megalin and the morphogen N-Shh. Specifically, we find that N-Shh binds to megalin with high

affinity and that the interaction is resistant to dissociation by low pH. We also show that one

consequence of the interaction is endocytosis of N-Shh. Megalin-mediated uptake of N-Shh

can be blocked by heparin, suggesting the involvement of heparan sulfate proteoglycans

(HSPGs) in the internalization process.

HSPGs have been implicated in N-Shh signaling (40, 41) and in the process of megalin-

mediated endocytosis of a number of its ligands (18). In the latter case, evidence suggests that

HSPGs serve to sequester ligands at or near the cell surface and thereby either facilitate

presentation of ligands to megalin or augment the affinity of ligands for megalin (18). Our

observation that N-Shh appeared to accumulate pericellularly on BN cells after blocking the

ligand-binding activity of megalin suggests the existence of an additional cell surface or

pericellular N-Shh-binding molecule. Considering recent evidence that Ptc is not detected at

significant levels on the cell surface (42), this other N-Shh binding component may very well be

HSPGs.

The likely significance of the interaction of N-Shh with megalin is that it impacts Shh

signaling. Three possibilities are that the interaction leads to: 1) direct signal transduction by

megalin, 2) modulation of the availability of N-Shh for its receptors or 3) transcytosis of N-Shh

important for long range N-Shh signaling. Direct signal transduction by megalin is supported by

recent evidence that other members of the LDLR family mediate signaling (43). For example,

LRP has been shown to interact with the heparin-binding growth factor, midkine, and regulate

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midkine-dependent survival of embryonic neurons (44). LRP has also been shown to interact

with PDGF-BB and function as a co-receptor in the process of PDGF signaling (45, 46).

Additionally, VLDLR and apoER2 interact with the neuronal protein reelin and mediate signaling

through the cyotplasmic adaptor protein Dab1 (47). With respect to the second possibility,

megalin-mediated endocytosis of N-Shh may modulate the extracellular levels of N-Shh and

thereby regulate availability to Ptc. For example, megalin might compete with Ptc for limiting

levels of N-Shh and thereby reduce Ptc dissociation from Smo leading to decreased Smo

signaling. Alternatively, megalin may deliver N-Shh to vesicular pools of Ptc and thus regulate

the potential of this Ptc to complex with Smo. The third possibility is consistent with the

emerging role of megalin as a mediator of transepithelial transport of various macromolecules.

For example, thyroglobulin (TG), transcobalamin-vitamin B12 complex and retinol-binding protein

(RBP) in complex with retinol/vitamin A are internalized by megalin, but avoid lysosomal

degradation and are delivered to the basolateral membrane from which they get released (48-

50). The mechanism by which megalin ligands bypass lysosomal degradation is not known.

One possibility is that the interaction between megalin and these ligands might not be readily

dissociated by acidic pH such as occurs in endocytic vesicles. As a consequence, the ligands

traffic together with the receptor and are either transported to apical or basolateral aspects of

the cell. Our finding that the megalin-N-Shh interaction is insensitive to low pH suggests that N-

Shh may also traffic in complex with megalin and thus be recycled and/or transcytosed. This

possibility is further supported by our findings from chloroquine experiments indicating that

endocytosed-N-Shh bypasses lysosomes.

Megalin-mediated transcytosis of N-Shh may facilitate long range signaling by N-Shh

during early development. For example, N-Shh expressed in the floor plate may bind to megalin

expressed on the apical surface of the neural tube epithelium and mediate transepithelial

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transport of N-Shh (Fig. 11). This process could account for delivery of N-Shh to cells in the

ventral region of the neural tube that undergo differentiation to form ventral nerves, a process

dependent on both N-Shh signal transduction and megalin expression (17, 19). A similar

process has been described in Drosophila involving transport of the morphogen Wingless

protein over large distances through imaginal disc epithelia (51). In this case, membrane

vesicles called argosomes, derived from the basolateral membranes, are transported

throughout imaginal disc epithelia. The argosomes are thought to originate from either

multivesicular endosomes and/or endosome transcytosis. Importantly, Wingless signaling has

been shown to involve a megalin family member, LRP6/arrow, although its exact role in the

process remains to be determined (52, 53).

In addition to the potential role of megalin in mediating long distance signaling via

transcytosis of N-Shh across the neural epithelium, its ability to endocytose N-Shh may also

impact N-Shh signaling in the early neural epithelium directly. Whether the mechanism for this

involves effects on the bioavailability of N-Shh or on the regulation of Ptc as described above,

the end result may be to influence N-Shh-dependent survival and differentiation of neural

epithelial cells (7, 8, 21). This hypothesis is supported by the megalin-deficient mouse

phenotype which demonstrates that megalin is required for normal viability and development of

the neuroepithelium (19).

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Acknowledgements

This work was supported by grants from the National Institutes of Health (HL61873) and the

American Heart Association, National (9950344N) to W.S.A. The authors thank Sandra Klatt for

technical assistance on this project, Drs. Paul Rayhorn and Kevin P. Williams (Biogen, Inc.,

Cambridge, MA) for technical advice concerning the differentiation of the C3H10T1/2 cells with

N-Shh and Dr. Waleed O. Twal for technical advice on BIAcore experimentation.

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Figure Legends

Figure 1. Megalin expression during early embryogenesis. Shown are confocal

micrographs of a 16-hour (A), a 24-hour (B) and a 33-hour (C-G) zebrafish embryo. In A,

megalin is expressed in the central region of the developing eyecup (arrowhead). At all stages,

megalin is expressed in the floorplate (arrows in A, B and D). At 24 h megalin is prominently

expressed in the floorplate and more dorsally in cells that line the lumen of the neural tube (B,

arrowhead). In a 33-hour embryo, megalin is extensively expressed on the lumenal surfaces of

the ventricles of the midbrain (mb) and forebrain (fb), with intense staining beginning at the

midbrain-hindbrain border (arrowhead, C and D). Megalin expression is intense on the interior

epithelial surface of the otic vesicle of the developing ear (E). Megalin is also expressed in the

paired pronephric ducts (arrowheads, F) of the forming kidney where it is also associated with

the lumenal side of the epithelium. A frontal view of the developing oral region (G) shows that

megalin is expressed in the ridge of the frontonasal process and maxillary processes

(arrowheads).

Figure 2. Comparative analysis of Shh and megalin expression during early

embryogenesis. Shown are confocal micrographs of lateral views of 19-hour (20 somite)

zebrafish embryos stained with antibodies to N-Shh (A) and megalin (B). Both proteins are co-

expressed in the floor plate of the brain (arrows). N-Shh is expressed in the notochord

(arrowhead, A) whereas megalin expression is not detectable in the notochord (arrowhead, B).

Cross sectional views of regions caudal to the hindbrain (insets) show that megalin is

expressed more dorsally in the neural tube whereas N-Shh is confined to the floor plate.

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Figure 3. Analysis of the integrity of recombinant GST-N-Shh. Panel A shows a Coomassie

staining of 5 µg of GST-N-Shh and GST. Panel B shows anti-N-Shh immunoblotting of 50 ng of

each protein. Panel C shows that GST-N-Shh is capable of stimulating10T1/2 cells to express

alkaline phosphatase (ALP), a marker of osteoblast differentiation. C3H10T1/2 cells were

treated for 5 days with 111 nM of commercial N-Shh, GST-N-Shh or GST and alkaline

phosphatase levels measured as described in Williams et al. (30).

Figure 4. ELISA and competitive radioligand binding assay demonstrate that N-Shh

binds to megalin and RAP inhibits the binding. In A, ELISA shows that both GST-N-Shh

(open circles) and commercially available N-Shh (filled circles) bind to megalin. In B,

homologous ligand displacement assay (open circles) was used to demonstrate the interaction

between [32P]-GST-N-Shh and megalin. In B, heterologous ligand displacement assay (filled

circles) was used to show that RAP inhibits binding of [32P]-GST-N-Shh to megalin. Curves

shown in B were based on fits of the data calculated using the computer program Ligand.

Figure 5. SPR analysis of Shh binding to megalin. Shown are SPR sensorgrams of GST-N-

Shh at varying concentrations (25-500 nM) binding to megalin immobilized on a sensor chip.

Data depicted is normalized to 100 response units and is representative of 6 separate

experiments. To obtain affinity constants (KD), SPR profiles in a given series were

simultaneously fit to a 1:1 binding site model using BIAevaluation 3.1 software.

Figure 6. N-Shh is endocytosed by BN cells and the uptake is inhibited by the megalin

antagonist RAP. In A, BN cells were incubated with GST-N-Shh or GST (20 nM) in the

presence of absence of RAP (1 µM) for 2 h and immunostained with anti-GST and FITC-

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labeled-anti-IgG (green). Nuclei were stained using TOTO-3 (blue). RAP treatment did not

effect binding of GST-N-Shh to the cell but inhibited its internalization. Shown are confocal

micrographs from two independent experiments. Panel B shows that megalin is the principal

RAP-binding protein present in detergent extracts of BN cells. Aliquots of BN cell extract were

immunoblotted with anti-megalin IgG (lane 1) or incubated with RAP (1 µM) and the bound RAP

then detected with mouse monoclonal anti-RAP IgG (lane 2). No other RAP-binding proteins

were evident even after prolonged exposure of the RAP overlay blot.

Figure 7. Megalin antagonists inhibit uptake of [32P]-GST-N-Shh by BN cells and

differentiated F9 cells. In A, BN cells were incubated for 2 h with [32P]-GST-N-Shh alone or in

the presence of RAP (1 µM), anti-megalin IgG (150 µg/ml), normal rabbit IgG (150 µg/ml).

In B, undifferentiated F9 cells (white bars) or F9 cells differentiated with retinoic acid and (black

bars) were incubated for 2 h with [32P]-GST-N-Shh alone or in the presence of RAP (1 µM).

Figure 8. Heparin inhibits uptake of [32P]-GST-N-Shh by BN cells and differentiated F9

cells. In A, BN cells were incubated for 2 h with [32P]-GST-N-Shh alone or in the presence of

heparin (1 µM). In B, undifferentiated F9 cells (white bars) or F9 cells differentiated with retinoic

acid and Bt2/cAMP (black bars) were incubated for 2 h with [32P]-GST-N-Shh alone or in the

presence of heparin (1 µM).

Figure 9. Chloroquine treament does not inhibit degradation of internalized N-Shh. BN

cells were incubated with 3 nM [32P]-GST-N-Shh (top panels) or 3 nM [32P]-GST-RAP (bottom

panels) alone or in the presence of RAP (1 µM), GST (1 µM) or chloroquine (0.1 mM).

Measurements of bound, internalized and degraded radiolabeled ligand were made after 3-h

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incubation. Note that RAP inhibits binding and internalization of [32P]-GST-N-Shh and [32P]-

RAP. By contrast, RAP and chloroquine both block degradation of labeled-RAP, but not of

labeled-N-Shh.

Figure 10. The N-Shh-megalin complex is resistant to dissociation by acidic pH. N-Shh

(A) or RAP (B) were allowed to associate for 2 min at neutral pH with megalin immobilized on a

sensor chip. The kinetics of N-Shh-megalin and RAP-megalin complex dissociation were

measured after the addition of protein-free buffer of neutral pH buffer or protein-free buffers of

acidic pH that ranged from 4.5 - 6.44. Shown are representative sensorgrams from experiments

that evaluated the effect of neutral pH and pH 4.5 buffers on the dissociation of each complex.

Arrows indicate the point of addition of protein-free flow buffer of indicated pH. Off rates (koff,

inset) were calculated using BIAevaluation 3.1 software.

Figure 11. Model of megalin-mediated transcytosis of N-Shh across neural tube

epithelial cells. Shown is a diagram of a neural tube in which cells of the ependymal layer

(green) transport N-Shh to Shh-responsive cells in the mantle layer (blue). N-Shh expressing

floorplate cells are red, lumenal epithelial cells are green, apical megalin expression is indicated

by purple and Shh-responsive cells are depicted in blue. Red arrows indicate the path of Shh

trafficking. The neural tube portion of the diagram was adapted from Balinsky (54).

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Scott ArgravesRobert A. McCarthy, Jeremy L. Barth, Mastan R. Chintalapudi, Christian Knaak and W.

Megalin functions as an endocytic sonic hedgehog receptor

published online April 18, 2002J. Biol. Chem. 

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